Gene transfer method

ABSTRACT

A method of transferring a foreign gene into cells, characterized by involving: the step of transferring into the cells with the use of an adenovirus vector, a first nucleic acid, which has a sequence provided with adeno-associated virus-origin ITRs in both sides of the target foreign gene to be transferred, and a second nucleic acid, which has an adeno-associated virus-origin rep gene and a promoter for expressing this gene and carries a stuffer sequence inserted thereinto sandwiched in two recombinase recognition sequences and located between the rep gene and the promoter; and the step of expressing the Rep protein under the action of recombinase in the cells obtained in the above step to thereby integrate the target foreign gene into the chromosomal DNA.

TECHNICAL FIELD

The present invention relates to a method that increases the efficiencyof gene transfer into target cells and enables efficient transformationof the target cells, as well as a series of techniques relatedtherewith, in the fields of medicine, cell technology, geneticengineering, developmental technology and the like.

BACKGROUND ART

Mechanisms of a number of human diseases have been elucidated. Therecombinant DNA techniques and the techniques for transferring a geneinto cells have rapidly progressed. Under these circumstances, protocolsfor somatic gene therapies for treating severe genetic diseases havebeen recently developed. More recently, attempts have been made to applythe gene therapy not only to treatment of the genetic diseases but alsoto treatment of viral infections such as AIDS as well as cancers.

Viral vectors currently used in general include retroviral vectors,adenoviral vectors and adeno-associated viral (AAV) vectors. Aretroviral vector can be readily prepared and integrate a foreign geneinto the chromosomal DNA of a target cell. Therefore, it is useful forgene therapy for which a long-term gene expression is desired. However,since a foreign gene is integrated at random sites in a chromosomal DNA,cancer or the like may be potentially caused if a retroviral vector isused. Furthermore, since a retroviral vector cannot infect cells inresting phase, the types of target cells are limited.

Although there were problems about the method for preparing adenoviralvectors, a convenient preparation method has been developed. The vectorcan efficiently infect many types of cells including cells in restingphase. However, since it does not have a mechanism for integrating aforeign gene into the chromosomal DNA of a target cell, the expressionof the foreign gene is usually transient.

An AAV vector can infect cells in resting phase and has a nature tointegrate a foreign gene specifically at the AAVS1 site on humanchromosome 19. Thus, it is expected that a gene transferred using theAAV vector is expressed for a long period in a cell without potentialrisk. However, there are practical problems associated with the AAVvectors that the preparation of AAV is complicated and the size of agene that can be transferred into cells is very small.

As described above, conventional viral vectors used for gene therapyhave their own advantages and disadvantages. No gene transfer methodthat enables convenient handling, high gene transfer efficiency andlong-term expression of a transferred gene was known. A system thatsatisfies these properties has been desired.

An attempt was made to develop such a system. In the system, a region ofAAV required for the site-specific integration of a foreign gene isincorporated into an adenoviral vector which has advantages of high genetransfer efficiency and easy preparation of a high-titer vector in orderto overcome the drawback of the adenoviral vectors that they cannotintegrate a foreign gene into the chromosomal DNA of a target cell (see,for example, U.S. Pat. No. 5,843,742).

However, since a Rep protein, which is encoded by the region of AAVrequired for the site-specific integration of a foreign gene, inhibitsthe proliferation of an adenovirus, an adenovirus having the regioncannot proliferate. Therefore, it is impossible to prepare such a vectorand, in consequence, gene transfer using such a vector is practicallyinfeasible.

Recchia et al. tried to solve the above-mentioned problem by using ahepatic cell-specific promoter, α1AT promoter, for expressing a rep gene(Recchia et al., Proceedings of the National Academy of Sciences of theUSA, 96:2615–2620 (1999)). Since the α1AT promoter is a hepaticcell-specific promoter, it does not operate in a virus-producer cell tobe used for the preparation of an adenoviral vector (a producer cell).Therefore, the rep gene contained in the adenoviral vector is notexpressed and the proliferation of the adenoviral vector is notinhibited.

However, the method of Recchia et al. has a drawback that the cell typein which a foreign gene can be integrated into the chromosomal DNA islimited to the hepatic cell because the promoter used for expressing therep gene is a hepatic cell-specific one.

The prior art has drawbacks as described above. A method which resultsin high gene transfer efficiency, by which a high-titer vector isreadily prepared, which enables the integration of a foreign gene intothe chromosomal DNA of a target cell, and which can be used to transfera foreign gene into a wide variety of cell types has been desired.

OBJECTS OF INVENTION

The main object of the present invention is to provide a gene transfermethod which results in high gene transfer efficiency, by which ahigh-titer vector is readily prepared, which enables the integration ofa foreign gene into the chromosomal DNA of a target cell, and which canbe used to transfer a foreign gene into a wide variety of cell types.

SUMMARY OF INVENTION

As a result of intensive studies, the present inventors have found thata foreign gene can be transferred into a wide variety of cell types andintegrated into the chromosomal DNAs by using an expression controlsystem that utilizes a recombinase and a recombinase recognitionsequence such as the Cre/loxP expression control system. Specifically,the present inventors have established a method and constructed a systemused for the method. In the method, the expression of a rep genecontained in an adenoviral vector in a virus-producer cell is repressedby inserting a stuffer sequence put between recombinase recognitionsequences. On the other hand, in the target cell having the adenoviralvector being transferred, the stuffer sequence put between therecombinase recognition sequences is removed through the action of arecombinase, the rep gene is expressed, and a foreign gene flanked byITRs transferred into the target cell can be integrated into thechromosomal DNA of the target cell. Thus, the present invention has beencompleted.

The present invention provides a method in which (1) a first nucleicacid which has a sequence in which ITRs from AAV are positioned on bothsides of a foreign gene of interest, and (2) a second nucleic acid inwhich a stuffer sequence put between two recombinase recognitionsequences is positioned between a rep gene from AAV and a promoter aretransferred into a cell using an adenoviral vector, and the foreign geneof interest is efficiently integrated into the chromosomal DNA of a widevariety of target cell types in a site-specific manner through theaction of a recombinase in the cell.

Specifically, when the above-mentioned two nucleic acids are transferredinto a target cell expressing a recombinase, the stuffer sequence putbetween two recombinase recognition sequences in the second nucleic acidis removed through the action of the recombinase in the target cell, andthe rep gene is expressed. The foreign gene in the first nucleic acid isintegrated into the chromosomal DNA of the target cell through theaction of a Rep protein expressed from the rep gene and the ITRs in thefirst nucleic acid.

The present invention is outlined as follows. The first aspect of thepresent invention relates to a method for transferring a foreign geneinto a cell, the method comprising:

-   -   (a) transferring into a cell using an adenoviral vector:        -   (1) a first nucleic acid which has a sequence in which ITRs            from adeno-associated virus are positioned on both sides of            a foreign gene of interest to be transferred; and        -   (2) a second nucleic acid which has a rep gene from            adeno-associated virus and a promoter for expressing the rep            gene and in which a stuffer sequence put between two            recombinase recognition sequences is inserted between the            rep gene and the promoter; and    -   (b) expressing a Rep protein in the cell obtained in step (a)        through the action of a recombinase to integrate the foreign        gene of interest into the chromosomal DNA of the cell.

The second aspect of the present invention relates to a system fortransferring a foreign gene into a cell, which contains:

-   -   (1) an adenoviral vector containing a first nucleic acid which        has a sequence in which ITRs from adeno-associated virus are        positioned on both sides of a gene of interest to be        transferred; and    -   (2) an adenoviral vector containing a second nucleic acid which        has a rep gene from adeno-associated virus and a promoter for        expressing the rep gene and in which a stuffer sequence put        between two recombinase recognition sequences is inserted        between the rep gene and the promoter.

The third aspect of the present invention relates to a system fortransferring a foreign gene into a cell, which contains an adenoviralvector containing:

-   -   a first nucleic acid which has a sequence in which ITRs from        adeno-associated virus are positioned on both sides of a gene of        interest to be transferred; and    -   a second nucleic acid which has a rep gene from adeno-associated        virus and a promoter for expressing the rep gene and in which a        stuffer sequence put between two recombinase recognition        sequences is inserted between the rep gene and the promoter.

The fourth aspect of the present invention relates to a transformed cellinto which a foreign gene is transferred by the method of the firstaspect.

The fifth aspect of the present invention relates to a transformed cellinto which a foreign gene is transferred using the system of the secondor third aspect.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: a figure illustrating the results of dot blot hybridization fordetecting site-specific integration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be explained in detail.

Adenoviruses are linear double-stranded DNA viruses. The size of thegenomic DNA is about 36 kb. A terminal protein encoded by the virus iscovalently bound to the 5′-termini on both ends of the genomic DNA toform a DNA-terminal protein complex (DNA-TPC).

There is no specific limitation concerning the adenovirus used as avector according to the present invention. For example, human adenovirustype 2 or 5 each exhibiting high proliferation efficiency and littlepathogenicity can be used. A nonproliferative adenovirus constructed bydeleting the E1 region involved in the replication of the virus from theadenoviral genome is used as a vector. Since an adenovirus lacking theE1 region lacks its competence to proliferate, proliferation of thevirus which may cause a disease state in a target cell does not occur.Therefore, such an adenovirus is preferable according to the presentinvention in view of safety.

There is no specific limitation concerning the deletion of a viralgenome as long as the resulting one can function as a vector. Anadenovirus from which the E3 region, which is not indispensable to theproliferation of the virus in cultured cells, is partially or entirelyremoved in addition to the above-mentioned E1 region can also be used.

An adenovirus lacking the E1 region or both the E1 region and the E3region can proliferate in the 293 cell derived from human embryonalkidney, which permanently expresses the genes in the E1 region.

In view of reduction in production of immunogens which may cause sideeffects, an adenovirus from which the E2 region and/or the E4 region isfurther deleted in addition to the E1 and E3 regions can also be used.Also, a gutless adenovirus vector (Parks et al., Proceedings of theNational Academy of Sciences of the USA, 93:13565–13570 (1996)) fromwhich the viral genes are fully removed can be used.

Such an adenovirus can proliferate in a cell that expresses the genescontained in the deleted region or upon coinfection with a helper virusthat contains the deleted region.

A first nucleic acid used according to the present invention is anucleic acid having a sequence in which inverted terminal repeats (ITRs)from adeno-associated virus (AAV) are positioned on both sides of aforeign gene of interest to be transferred. Although it is not intendedto limit the present invention, the first nucleic acid is preferablyused being incorporated in an adenoviral vector. The gene of interestcontained in this nucleic acid is integrated into the genome of a targetcell in a site-specific manner through the action of the ITRs from AAVand a Rep protein expressed from a second nucleic acid described below.

ITRs are T-shaped hairpin structures each consisting of 145 baseslocated on both ends of the single-stranded DNA of AAV. They containsequences indispensable to integration of the viral genome at the AAVS1site on chromosome 19 of human as its host (Yukihiko Hirai, Jikken Igaku(Experimental Medicine), 12 (15):1811–1816 (1994)). ITRs used accordingto the present invention are not specifically limited as long as theyhave activities of integrating at the AAVS1 site, and may be subjectedto substitution, deletion, insertion or addition.

There is no specific limitation concerning a foreign gene to be insertedbetween two ITRs. It may be any gene of which the transfer is desired.Genes encoding enzymes or other proteins connected with diseases to betreated, and genes encoding functional nucleic acid molecules such asantisense nucleic acids, ribozymes or decoys are exemplified. There isalso no specific limitation concerning the origin of the gene. The genemay be one derived from an organism species that is the same as ordifferent from that of the cell into which the gene is to betransferred, chemically synthesized one, or a combination of thereof.Appropriate regulatory elements such as a promoter and an enhancer forregulating the expression of the gene may be added to the gene.

Although it is not intended to limit the present invention, the desiredsize of the foreign gene of interest to be inserted between two ITRs is,for example, in case of one lacking the E1 and E3 regions, about 7.2 kbor less in view of packaging of a recombinant adenoviral vector. In caseof a gutless adenovirus vector, the size is desirably 37 kb or less.

The promoter to be used for the first nucleic acid according to thepresent invention is not specifically limited as long as it can expressa gene of interest in a target cell. For example, such promoters includeCAG promoter, SRα promoter, EF1α promoter, CMV promoter and PGKpromoter, and can selected depending on the purpose. If atissue-specific promoter is used, a gene of interest can be expressed ina tissue-specific manner. For example, expression can be conducted in ahepatic cell-specific manner by using liver-specific α1AT promoter, in askeletal muscle-specific manner by using skeletal muscle-specificα-actin promoter, in a nerve-specific manner by using nerve-specificenolase promoter, in a vascular endothelial cell-specific manner byusing vascular endothelial cell-specific tie promoter, in a gastriccancer-specific manner by using gastric cancer-specific CEA promoter, orin a hepatic cancer-specific manner by using hepatic cancer-specific AFPpromoter.

A second nucleic acid according to the present invention is one in whicha promoter/a recombinase recognition sequence/a stuffer sequence/arecombinase recognition sequence/a rep gene from AAV are arranged inthis order. Although it is not intended to limit the present invention,the second nucleic acid is preferably used being incorporated in anadenoviral vector. This nucleic acid expresses a rep gene in a targetcell. The rep gene is required for the integration of a foreign gene ofinterest contained in the above-mentioned first nucleic acid into thechromosomal DNA of the target cell.

The rep gene, which has cytotoxicity and an activity of inhibitingproliferation of an adenovirus, is not expressed in a cell for theproliferation of an adenovirus (a producer cell) which does not containa recombinase. This is because a stuffer sequence exists between thepromoter and the rep gene in this nucleic acid. Thus, it can be used toproliferate the virus in large quantities. On the other hand, thestuffer sequence put between the recombinase recognition sequences isexcised through the action of a recombinase in any target cellexpressing the recombinase. As a result, the promoter is placed adjacentto the initiation codon of the rep gene, enabling the expression of therep gene.

The Rep region of AAV encodes four proteins, Rep78, Rep68, Rep52 andRep40. Among these, Rep78 protein or Rep68 protein (called large Rep) isindispensable to the integration of the AAV genome at the AAVS1 site onhuman chromosome 19. The rep gene used according to the presentinvention is not specifically limited as long as it has an activity ofintegrating a foreign gene of interest contained in the nucleic acid ofthe first aspect into the chromosomal DNA of a target cell. For example,it may be one encoding Rep78 protein, Rep68 protein, or a proteinsubjected to substitution, deletion, addition or insertion in Rep78protein or Rep68 protein without losing the activity.

The region encoding Rep78 protein and Rep68 protein also encodes Rep52protein and Rep40 protein. Rep52 protein and Rep40 protein haveactivities of inhibiting the proliferation of an adenovirus. Thus, it isdesirable to modify the rep gene used according to the present inventionby site-directed mutagenesis or the like so as to prevent the expressionof Rep52 protein and Rep40 protein. For example, the expression of Rep52protein and Rep40 protein can be prevented by changing the 225th aminoacid residue in Rep78 protein from methionine to glycine.

There is no specific limitation concerning a promoter to be used for thesecond nucleic acid according to the present invention as long as it canexhibit its activity in a target cell and express a rep gene afterremoval of a stuffer sequence. The promoter can be selected depending onthe purpose. The promoter used for the second nucleic acid can beselected regardless of its expression in a producer cell because theexpression of the promoter is almost completely repressed in theproducer cell due to the existence of the stuffer sequence.

For example, p5 promoter from AAV, SRα promoter, EF1α promoter, CMVpromoter, SV40 promoter, a promoter from a virus such as 5′ LTR promoterfrom murine leukemia virus, and a fused promoter having a portion from avirus such as CAG promoter are known to operate in a number of celltypes regardless of tissues. Therefore, they are preferable if many celltypes are to be targeted.

Furthermore, a gene of interest can be transferred in a tissue-specificmanner by selecting a tissue-specific promoter. There is no specificlimitation concerning the tissue or the cell into which a gene ofinterest is to be transferred as long as an adenovirus can infect it andthe AAVS1 site is present on its chromosome. A gene of interest can betransferred into substantially all human tissues or cells by selecting apromoter at will. For example, skeletal muscle-specific α-actinpromoter, nerve-specific enolase promoter or vascular endothelialcell-specific tie promoter can be selected if the gene of interest is tobe transferred into skeletal muscle, nerve or vascular endothelialcells, respectively. In addition, if a suicide gene is to be transferredtargeting a cancer cell, gastric cancer-specific CEA promoter, hepaticcancer-specific AFP promoter or the like may be selected.

In the system of the present invention, as the expression of a rep geneis increased, the efficiency of integration of a foreign gene ofinterest into a chromosomal DNA may be increased. However, on the otherhand, it is considered that the toxicity against a target cell may alsobe increased. Since the toxicity of the product of the rep gene, the Repprotein, to a cell varies depending on the type of the cell, it isnecessary to optimize the expression level of the rep gene depending onthe target cell. For this purpose, a promoter having an optimalexpression strength for the target cell may be selected. For example, itis known that the expression strengths of the above-mentioned CAG, CMVand SV40 promoters in many cell types are as follows: CAG promoter>CMVpromoter>SV40 promoter. Thus, for example, SV40 promoter, CAG promoteror CMV promoter can be used if a cell type that exhibits high, low ormoderate sensitivity to the cytotoxicity of the Rep protein is used as atarget cell, respectively.

Furthermore, the expression of p5 promoter from AAV is repressed in thepresence of the Rep protein. If a promoter like p5 promoter is used,once the rep gene is expressed, the activity of the promoter is thenrepressed by the Rep protein expressed from the rep gene. As a result,overexpression of the rep gene is repressed. Thus, the toxicity due tothe Rep protein can be limited to a minimum.

A stuffer sequence used according to the present invention is a sequencethat intervenes between a promoter and a rep gene in a second nucleicacid to repress the expression of the rep gene from the promoter. Thereis no specific limitation concerning the stuffer sequence as long as itintervenes between the promoter and the rep gene to repress theexpression of the rep gene. However, it desirably contains a terminatorsequence, a poly(A) sequence or the like in view of strict expressioncontrol. Since the expression from the promoter used for the secondnucleic acid is almost completely repressed by the stuffer sequence, itis expected that a recombinant adenovirus is efficiently produced in aproducer cell. The repression of the promoter due to the stuffersequence in the producer cell can be confirmed at the level oftranscription (e.g., using Northern blotting or RT-PCR), or bydetermining the ability of the producer cell to produce recombinantviruses.

Expression control systems that utilize recombinases and recombinaserecognition sequences include the following: Cre/loxP expression controlsystem (Kanegae et al., Nucleic Acids Research, 23:3816–3821 (1995)); anexpression control system that utilizes a recombinase encoded by FLPgene from yeast 2μ plasmid (Broarch et al., Cell, 29:227–234 (1982));and an expression control system that utilizes a recombinase encoded byR gene from Zygosaccharomyces rouxii pSR1 plasmid (Matsuzaki et al.,Molecular and Cellular Biology, 8:955–962 (1988)). Although it is notintended to limit the present invention, the expression control systemused according to the present invention is preferably the Cre/loxPexpression control system in view of recombination efficiency.

There is no specific limitation concerning the recombinase recognitionsequence to be used according to the present invention as long as arecombinase can recognize it to remove a stuffer sequence put betweenthe sequences. The recombinase recognition sequence is exemplified bythe loxP sequence which is a sequence recognized by the recombinase Crein the Cre/loxP expression control system.

The loxP sequence is a sequence recognized by Cre, a site-specificrecombinase encoded by Escherichia coli P1 phage (Abremski et al.,Journal of Biological Chemistry, 259:1509–1514 (1984), Hoess et al.,Proceedings of the National Academy of Sciences of the USA, 81:1026–1029(1984), Hoess et al., Journal of Molecular Biology, 181:351–362 (1985)).The recombinase Cre specifically recognizes a minimal unit in the loxPsequence consisting of 34 bases to effect cleavage of DNA and stranddisplacement between two loxP sequences. Specifically, if two loxpsequences exist on the same molecule in the same direction, a DNAsequence put between the loxP sequences is excised as a circularmolecule through the action of the recombinase Cre (an excisionreaction). Only the minimal unit as described in Hoess et al(Proceedings of the National Academy of Sciences of the USA,81:1026–1029 (1984)) may be used as a loxP sequence according to thepresent invention.

There is no specific limitation concerning the loxP sequence usedaccording to the present invention as long as the recombinase Crerecognizes it to effect an excision reaction. It may be the wild-typeloxP sequence from Escherichia coli P1 phage or a mutant sequencesubjected to substitution, deletion, addition or insertion to the extentthat the recombinase Cre recognizes it to effect an excision reaction.Such a mutant sequence is exemplified by loxP2272, loxP5171, loxP2271,loxP3171, loxP5272 or loxP5372 as described in Lee et al., Gene,216:55–65 (1998).

There is no specific limitation concerning the recombinase usedaccording to the present invention. A recombinase that can recognize arecombinase recognition sequence in a second nucleic acid to effect anexcision reaction may be selected and used. For example, a site-specificrecombinase from Escherichia coli P1 phage, Cre, can be used to excise astuffer sequence put between loxP sequences. The recombinase usedaccording to the present invention is not specifically limited as longas it recognizes a recombinase recognition sequence to exhibit a DNArecombination activity. For example, it may be one having an amino acidsequence of a wild-type recombinase subjected to substitution, deletion,addition or insertion to the extent that it exhibits the activity.Alternatively, one to which a nuclear transport signal is added forfacilitating nuclear transport in a cell may be used.

There is no specific limitation concerning the method for expressing arecombinase in a target cell. For example, a nucleic acid encoding arecombinase can be transferred into a cell for expression. Although themethod for transferring such a nucleic acid is not specifically limited,a transfer method using an adenoviral vector is preferable because theadenoviral vector results in high gene transfer efficiency and ahigh-titer vector can be readily prepared for it.

There is no specific limitation concerning the promoter used for avector for expressing a recombinase as long as it can express arecombinase gene to induce the expression of Rep and enable theintegration of a foreign gene of interest. For example, it may be apromoter having an activity in various tissues such as CAG promoter, SRαpromoter, EF1α promoter, CMV promoter or PGK promoter. Alternatively, itmay be a tissue-specific promoter which operates only in a specifictissue such as the above-mentioned liver-specific α1AT promoter. If atissue-specific promoter is used, it is possible to specificallyintegrate a foreign gene only into the chromosomal DNAs of cells in aspecific tissue.

An adenoviral vector containing a first nucleic acid and an adenoviralvector containing a second nucleic acid may be transferred into a cellexpressing a recombinase in order to transfer a gene of interest into atarget cell. The two vectors may be transferred simultaneously orseparately. Alternatively, a single adenoviral vector containing boththe first and second nucleic acids may be transferred into a cell.Specifically, an adenoviral vector containing the elements of the firstnucleic acid (a gene of interest put between ITRs from AAV) and theelements of the second nucleic acid (a promoter/a recombinaserecognition sequence/a stuffer sequence/a recombinase recognitionsequence/a rep gene from AAV) on a single vector may be transferred.

If a recombinase is to be expressed from a recombinase gene transferredinto a cell, an adenoviral vector containing a first nucleic acid, anadenoviral vector containing a second nucleic acid, and a vector fortransferring the recombinase gene into a cell may be transferred intothe target cell. The three vectors may be transferred simultaneously orseparately. The first and second nucleic acids may be contained in asingle adenoviral vector. In this case, this vector is used incombination with a vector for transferring a recombinase gene into acell. Alternatively, the first nucleic acid and the recombinase gene maybe contained in a single adenoviral vector. In other words, anadenoviral vector constructed to contain the elements of the firstnucleic acid (a gene of interest put between ITRs from AAV) and arecombinase gene in a single vector, and an adenoviral vector containingthe second nucleic acid may be used.

A recombinase (e.g., the recombinase Cre) expressed in a target cellrecognizes two recombinase recognition sequences (e.g., the loxPsequences) in a second nucleic acid, and removes a stuffer sequence putbetween the two recombinase recognition sequences through an excisionreaction. As a result, a promoter in the second nucleic acid is placedadjacent to a rep gene, resulting in the expression of the rep gene bythe action of the promoter. A gene of interest put between ITRs in afirst nucleic acid is integrated at the AAVS1 site on chromosome 19 ofthe target cell by the action of a Rep protein produced from the secondnucleic acid. The gene of interest integrated into the chromosome of thetarget cell as described above is stably retained in the target cell andstably expressed for a long period. Furthermore, since any promoter canbe used as a promoter for expressing a rep gene, a gene of interest canbe expressed in any cell.

One can be readily prepared a high-titer virus for the adenoviral vectorused according to the present invention like for conventionaladenoviruses. The method for preparing an adenoviral vector according tothe present invention is exemplified by the COS-TPC method (Proceedingsof the National Academy of Sciences of the USA, 93:1320 (1996)). Usingthis method, a vector according to the present invention can beefficiently prepared without complicated procedures.

An adenoviral vector used according to the present invention canefficiently infect a number of cell types including those in restingphase like an adenovirus. Therefore, it can be utilized for genetherapies for various tissues or cells. Furthermore, since it canintegrate a foreign gene in a site-specific manner like AAV, it can beused to express the foreign gene for a long period without the risk ofdeveloping cancer or the like due to integration at random sites.

Furthermore, unlike retroviral vectors, a foreign gene of interest canbe expressed using any promoter for the adenoviral vector to be usedaccording to the present invention. Since a rep gene can also beexpressed using any promoter, there is no specific limitation concerningthe cell type to which the present invention can be applied, and theforeign gene can be transferred into any type of target cell. Inaddition, unlike AAV, a large-sized foreign gene of interest can beinserted.

Thus, the gene transfer method of the present invention is an excellentsystem which overcomes all the drawbacks associated with conventionalsystems including limited target cell type, inefficient infection, shortexpression period and difficult expression control. Thus, it can be usedfor all gene therapies whose effects were limited due to the limitationof conventional methods and remarkable therapeutic effects are expected.

There is no specific limitation concerning the disease to be subjectedto gene therapy using the gene transfer method of the present invention.The method can be utilized to treat a genetic disease for which acongenital genetic abnormality is observed, a viral infection such asAIDS, and cancer. Since a gene transferred using the gene transfermethod of the present invention is integrated into the chromosomal DNAof a target cell, it is possible to express a foreign gene for a longperiod. Thus, the method is particularly useful for treatment of agenetic disease for which a congenital genetic abnormality is observedsuch as adenosine deaminase deficiency, muscular dystrophy orphenylketonuria.

Furthermore, the gene transfer method of the present invention is usefulnot only for gene therapy but also for obtaining cells having variousforeign genes in vitro. The thus obtained cells having genes beingtransferred are useful for production of useful substances, developmentof disease models and the like.

Furthermore, one can conveniently carry out the present invention byusing a kit containing an adenoviral vector having a first nucleic acidand an adenoviral vector containing a second nucleic acid according tothe present invention. For example, the kit contains a component forconstructing an adenoviral vector having a first nucleic acid into whicha selected foreign gene of interest is incorporated, and an adenoviralvector containing a second nucleic acid. A vector for expressing arecombinase may be further included in the kit. Additionally, a culturedcell for preparing an adenovirus (a producer cell), a medium, a cellculture plate and the like may be included.

The gene transfer method of the present invention can be carried out,for example, as follows.

An adenoviral vector containing a first nucleic acid can be prepared asfollows.

The rep region and the cap region are removed from a plasmid containinga genomic DNA from AAV, and a foreign gene to be transferred is insertedinto the resulting product. The source of the foreign gene to betransferred is not specifically limited. It may be prepared from agenome or a recombinant, amplified by a PCR, or chemically synthesized.The plasmid prepared as described above has an integration unit thatconsists of a sequence in which ITR sequences from AAV are positioned onboth sides of the foreign gene.

Next, the integration unit is prepared from the resulting plasmid bydigestion with a restriction enzyme. The integration unit is insertedinto a cosmid pAxcw (a cosmid containing almost entire adenoviral genomelacking the E1 and E3 genes) contained in Adenovirus Expression VectorKit (Takara Shuzo, hereinafter referred to as Kit 1) at its SwaI site. Arecombinant adenovirus that contains the integration unit (a firstnucleic acid) can be prepared from the resulting recombinant cosmidusing Kit 1.

An adenoviral vector containing a second nucleic acid can be prepared asfollows.

A region encoding Rep78 protein is prepared from the plasmid containingthe genomic DNA from AAV. The region encoding Rep78 protein can beobtained by PCR amplification using the above-mentioned plasmid as atemplate or treatment of the plasmid with a restriction enzyme.

A region encoding Rep52 protein which represses the proliferation of anadenovirus is contained in the DNA fragment obtained as described above.Then, the nucleotide sequence for the 225th amino acid residue in Rep78protein is changed from ATG which corresponds to methionine to GGA whichcorresponds to glycine using site-directed mutagenesis in order toeliminate the expression from that region.

Next, the mutant rep 78 gene fragment is inserted into a cosmidpAxCALNLw that contains an adenoviral genome, a promoter/a loxPsequence/a stuffer sequence/a loxP sequence/a SwaI cloning site at theSwaI site to prepare a cosmid containing a promoter/a loxP sequence/astuffer sequence/a loxP sequence/a mutant rep78 gene fragment (a secondnucleic acid). An adenovirus that contains a promoter/a loxP sequence/astuffer sequence/a loxp sequence/a mutant rep78 gene fragment (a secondnucleic acid) can be prepared from the resulting cosmid using Kit 1.

A cosmid pAxCALNLw attached to Adenovirus Cre/loxP-Regulated ExpressionVector Kit (Takara Shuzo, hereinafter referred to as Kit 2) can be usedfor the preparation of an adenovirus containing a second nucleic acid.It is prepared by inserting, in this order, a loxP sequence, a neomycinresistance gene, an SV40 virus poly(A) signal, a loxP sequence and aSwaI linker into pAxCAwt attached to Kit 1 at its SwaI site. pAxCAwt isa cosmid prepared by inserting CAG promoter sequence into theabove-mentioned cosmid pAxcw. In this case, the neomycin resistance geneand the SV40 virus poly(A) signal correspond to a stuffer sequence.

An adenoviral vector for expressing cre gene can be prepared using Kit 1from a recombinant cosmid which is obtained by inserting the cre geneinto the cosmid pAxcw at the SwaI site as described for the first andsecond vectors. Alternatively, an adenoviral vector for expressing thecre gene, AxCANCre, as described in Kanegae et al., Nucleic AcidsResearch, 23:3816–3821 (1995) may be used. The adenoviral vectorAxCANCre attached to Adenovirus Cre/loxP-Regulated Expression Vector Kit(Takara Shuzo, Kit 2) may be used. A method for determining the titer ofa recombinant adenovirus obtained as described above is known in theart. For example, the titer can be determined according to the method ofKit 1.

The three types of adenoviral vectors obtained as described above aretransferred into a target cell according to a conventional method. Inthe cell, a recombinase Cre generated as a result of the expression ofthe cre gene removes the stuffer sequence put between the loxPsequences. Subsequently, Rep protein generated as a result of theexpression of the rep gene integrates the foreign gene of interestpositioned between the ITRs into the chromosomal DNA of the cell. It ispossible to confirm if the foreign gene of interest is integrated at theAAVS1 site in the chromosomal DNA of the target cell in a site-specificmanner by carrying out a PCR. In the PCR, a chromosomal DNA preparedfrom the target cell for gene transfer is used as a template, and aprimer that anneals to the integration unit and a primer that anneals tothe AAVS1 site are used.

The frequency of specific integration in unit cell number (integrationefficiency) can be determined, for example, according to the followingmethod.

A first nucleic acid in which a drug resistance gene (e.g., a neomycinresistance gene) is inserted in the integration unit is prepared. Ifthis first nucleic acid is integrated into the chromosomal DNA of atarget cell, daughter cells generated as a result of proliferation ofthe target cell exhibit drug resistance because they always have thedrug resistance gene. On the other hand, if the integration into thechromosomal DNA of the target cell does not occur, the daughter cellsgenerated as a result of proliferation of the target cell become drugsensitive because the drug resistance gene transferred into the cell isnot retained in the daughter cells. Thus, when the target cell iscultured in a medium containing a drug (e.g., neomycin) for a longperiod after transferring the first nucleic acid having the drugresistance gene being inserted into the target cell, only cells in whichintegration has been occurred proliferate and form colonies (drugresistant colonies).

The efficiency of integration of a foreign gene into a target cell canbe determined as a ratio of the number of drug resistant colonies (Drugresistant) to the number of colonies formed after culturing for a longperiod in a medium without a drug (Total) according to the followingequation:(Integration efficiency)=(Drug resistant)+(Total).

It should be noted that the integration efficiency determined asdescribed above includes that of non-site-specific integration. Theefficiency of site-specific integration can be determined as follows:several drug resistant colonies are picked up; specific integration isconfirmed by the above-mentioned method using a PCR; a ratio of colonieswith site-specific integration in drug resistant colonies is determined;the efficiency of site-specific integration of a foreign gene into atarget cell is then determined by multiplying the above-mentionedintegration efficiency by the determined ratio.

EXAMPLES

The following Examples further illustrate the present invention indetail but are not to be construed to limit the scope thereof.

Example 1 Preparation of Recombinant Adenoviral Vector

1.1 Preparation of first vector (adenoviral vector containing firstnucleic acid)

An adenoviral vector AxAAVZ that contains β-galactosidase gene as aforeign gene was prepared as follows.

A plasmid pAV1 (ATCC 37215) contains a genomic DNA from AAV having anucleotide sequence of SEQ ID NO:1. A double-stranded EcoRV linkerconsisting of a synthetic DNA having a nucleotide sequence of SEQ IDNO:4 and a synthetic DNA having a nucleotide sequence of SEQ ID NO:5 wasinserted into pAV1 at the AvaII site (nucleotide number 191 in SEQ IDNO:1). A double-stranded EcoRV linker consisting of two synthetic DNAstrands each having a nucleotide sequence of SEQ ID NO:6 being annealedeach other was further inserted into pAV1 at the NcoI site (nucleotidenumber 4485 in SEQ ID NO:1). As a result, a plasmid pAV1E5 was obtained.A DNA fragment (βgal expression unit) which comprises CMV promoter,β-galactosidase gene and SV poly(A) signal was obtained by digesting aplasmid pβgal (Clontech) with EcoRI and HindIII. After blunting the endsof this fragment using DNA Blunting Kit (Takara Shuzo), the resultingfragment was inserted into pAV1E5 at its EcoRV site. The thus obtainedplasmid was digested with BglII to prepare the βgal expression unit putbetween ITRs from AAV (hereinafter referred to as an integration unit).The integration unit was inserted into a cosmid pAxcw contained inAdenovirus Expression Vector Kit (Takara Shuzo, Kit 1) at its SwaI site.A recombinant adenovirus AxITRZ was prepared from the resultingrecombinant cosmid using Kit 1.

1.2 Preparation of second vector (adenoviral vector containing secondnucleic acid)

An adenoviral vector AxCALNLRep78 for expressing a rep gene in a targetcell was prepared as follows.

A PCR was carried out using the plasmid pAV1 as a template as well as aprimer REPF having a nucleotide sequence of SEQ ID NO:2 and a primerREPR having a nucleotide sequence of SEQ ID NO:3 to amplify a region ofnucleotide numbers from 313 to 2205 in SEQ ID NO:1 which contained rep78gene. The PCR was carried out using TaKaRa Taq (Takara Shuzo).

The resulting amplified DNA fragment contained rep52 gene whichrepresses proliferation of an adenovirus in addition to the rep78 gene.The 225th amino acid residue in Rep78 was changed from methionine toglycine in order to eliminate the expression of this gene. Specifically,ATG at nucleotide numbers 993–995 in SEQ ID NO:1 was changed to GGAusing the plasmid pAV1 as a template, an oligonucleotide having anucleotide sequence of SEQ ID NO:11 and Mutan Super Expression Km(Takara Shuzo). An amplified DNA fragment was then obtained as describedabove.

The mutant amplified DNA fragment was inserted into a cosmid pAxCALNLwattached to Adenovirus Cre/loxP-Regulated Expression Vector Kit (TakaraShuzo, Kit 2) at its SwaI site. Then, an adenovirus AxCALNLRep78 wasprepared using Kit 1.

1.3 Third vector (vector for expressing cre gene)

An adenoviral vector AxCANCre attached to Adenovirus Cre/loxP-RegulatedExpression Vector Kit (Takara Shuzo, Kit 2) was used for expressing cregene.

Example 2

Infection Experiments

HeLa cells (ATCC CCL-2) were cultured in a 24-well cell culture plate(Iwaki Glass). DMEM medium containing 10% fetal calf serum (both fromBio Whittaker) was used for culturing the cells. Infections were carriedout according to the instructions attached to Kit 1 using one of therecombinant adenoviruses AxCANCre, AxCALNLRep78 and AxITRZ alone or acombination thereof. After culturing for two days, insertion of theintegration unit at the AAVS1 site in an infected cell was examined asfollows.

A genomic DNA was prepared from an infected cell according to the methodas described in the instructions attached to Kit 1. A PCR was carriedout using the resulting genomic DNA as a template as well as a primer 81(SEQ ID NO:7) which anneals to the integration unit and a primer 1722(SEQ ID NO:8) which anneals to the AAVS1 site. One μl each of the PCRproducts was spotted onto a nylon membrane (Hybond-N, Amersham). Dotblot hybridization was carried out using DIG Labeling & Detection Kit(Boehringer-Mannheim) to detect site-specific integration. A probe wasprepared using a human genomic DNA as a template, primers 79 and 80having nucleotide sequences of SEQ ID NOS:9 and 10, respectively, andPCR DIG Probe Synthesis Kit (Boehringer-Mannheim). The resulting probehas the nucleotide sequence of the AAVS1 site. The results are shown inFIG. 1. As shown in FIG. 1, site-specific integration of the foreigngene did not occur when infections were carried out using one or two ofthe three recombinant viruses AxCANCre, AxCALNLRep78 and AxITRZ.Site-specific integration was observed only in the cell co-infected withthe three recombinant viruses.

Example 3

Next, infection experiments were carried out using a human lung-derivedcell line A549 (ATCC CCL-185) or a human liver-derived cell line HepG2(ATCC HB-8065) as a target cell to examine transfer of a foreign geneinto a cell other than the HeLa cell.

Infections were carried out as described in Example 2 using one of therecombinant adenoviruses AxCANCre, AxCALNLRep78 and AxITRZ obtained inExample 1 alone or a combination thereof. Site-specific integration wasdetected according to the method as described in Example 2.

As a result, for both the A549 cell and the HepG2 cell, site-specificintegration of the foreign gene did not occur when infections werecarried out using one or two of the three recombinant viruses AxCANCre,AxCALNLRep78 and AxITRZ, and was observed only in the cell co-infectedwith the three recombinant viruses as observed for the HeLa cell.

These results suggest that a foreign gene can be integrated into notonly the HeLa cell but also various human cells in a site-specificmanner according to the method of the present invention.

INDUSTRIAL APPLICABILITY

The present invention provides a method for transferring a foreign genein which the foreign gene is efficiently transferred into a human cellor a human individual and integrated specifically at the AAVS1 site onchromosome 19. The present invention is particularly useful for genetherapy.

Sequence Listing Free Text

SEQ ID NO:2: PCR primer designated as REPF to amplify Rep78 gene.

SEQ ID NO:3: PCR primer designated as REPR to amplify Rep78 gene.

SEQ ID NO:4: EcoRV linker.

SEQ ID NO:5: EcoRV linker.

SEQ ID NO:6: EcoRV linker.

SEQ ID NO:7: PCR primer designated as 82 which anneals with integrationunit.

SEQ ID NO:8: PCR primer designated as 1722 which anneals with AAVS1region.

SEQ ID NO:9: PCR primer designated as 80 to amplify AAVS1 region used asa probe.

SEQ ID NO:10: PCR primer designated as 80 to amplify AAVS1 region usedas a probe.

SEQ ID NO:11: Designed oligonucleotide to introduce mutation into Rep78gene.

1. A system for transferring a foreign gene into a cell, which contains:a. a combination of:
 1. an adenoviral vector containing a first nucleicacid which has a sequence in which ITRs from adeno-associated virus arepositioned on both sides of a gene of interest to be transferred; and 2.an adenoviral vector containing a second nucleic acid which has a repgene from adeno-associated virus in which the expression of Rep52protein and Rep40 protein are prevented by changing the 225^(th) aminoacid residue in Rep78 from methionine to glycine and a promoter forexpressing the rep gene and in which a stuffer sequence put between tworecombinase recognition sequences is inserted between the rep gene andthe promoter; or b. an adenoviral vector containing: a first nucleicacid which has a sequence in which ITRs from adeno-associated virus arepositioned on both sides of a gene of interest to be transferred; and asecond nucleic acid which has a rep gene from adeno-associated virus anda promoter for expressing the rep gene and in which a stuffer sequenceput between two recombinase recognition sequences is inserted betweenthe rep gene and the promoter.
 2. The system according to claim 1, whichcontains a vector for transferring a recombinase gene into a cell. 3.The system according to claim 2, wherein the vector for transferring arecombinase gene into a cell is an adenoviral vector.
 4. The systemaccording to claim 3, wherein the adenoviral vector containing the firstnucleic acid, the adenoviral vector containing the second nucleic acidand the vector for transferring a recombinase gene into a cell aredifferent each other.
 5. The system according to claim 1, wherein therecombinase recognition sequences in the second nucleic acid are loxPnucleotide sequences, and the recombinase is Cre, a recombinase fromEscherichia coli P1 phage.
 6. A transformed cell into which a foreigngene is transferred using the system defined by claim
 1. 7. A system fortransferring a foreign gene into a cell, which system contains acombination of: a. an adenoviral vector containing a first nucleic acidwhich has a sequence in which ITRs from adeno-associated virus arepositioned on both sides of a gene of interest to be transferred and arecombinase gene; and b. an adenoviral vector containing a secondnucleic acid which has a rep gene from adeno-associated virus and apromoter for expressing the rep gene and in which a stuffer sequence putbetween two recombinase recognition sites is inserted between the repgene and the promoter.